The activity of the P-glycoprotein (P-gp) transporter encoded by the ABCB1 gene confers resistance to anticancer drugs and contributes to cancer-related mortality and morbidity. Recent studies revealed the cytotoxic effects of the endogenous dipeptide carnosine. The current study aimed to investigate the role of carnosine as a potential inhibitor of P-gp activity. We used molecular docking and molecular dynamic simulations to study the possible binding and stability of carnosine-P-gp interactions compared with verapamil. In vitro assays using doxorubicin-resistant NCI/ADR-RES cells were established to test the effects of carnosine (10–300 µM) on P-gp activity by the rhodamine-123 efflux assay and its effect on cell viability and doxorubicin-induced cytotoxicity. Verapamil (10 µM) was used as a positive control. The results showed that carnosine binding depends mainly on hydrogen bonding with GLU875, GLN946, and ALA871, with a higher average Hbond than verapamil. Carnosine showed significant but weaker than verapamil-induced rhodamine-123 accumulation. Carnosine and verapamil similarly inhibited cell viability. However, verapamil showed a more significant potentiating effect on doxorubicin-induced cytotoxicity than a weaker effect of carnosine at 300 µM. These results suggest that carnosine inhibits P-gp activity and potentiates doxorubicin-induced cytotoxicity at higher concentrations. Carnosine might be a helpful lead compound in the fight against multidrug-resistant cancers. According to the WHO records, cancer ranks second as a cause of death worldwide, with a death toll of 10 million in 2018 alone . These tThe activity of the ABC family of transporters protects normal body cells from chemical insults by shuttling xenobiotic molecules across the cell membrane . ContrarWith the current understanding of the domain structure of P-gp ,17, in sDocking studies were performed to assess the binding properties of carnosine with P-gp. The standard Glide docking protocol (SP) was used. Carnosine showed a docking score of −5.54, compared with −7.38 and −9.1 for verapamil and the co-crystalized ligand (paclitaxel), respectively. These values reflect the interactions of each compound with P-pg, which is affected by the size and volume of interactions of each compound with the protein. In terms of ligand efficiency, measured as the binding energy normalized to the number of atoms in the ligand, carnosine showed the most favorable binding profile, evidenced by the lowest ligand efficiency values . In addiThe average change in displacement of a group of atoms with respect to a reference frame is calculated in terms of the RMSD. During the simulation, monitoring the protein’s RMSD can provide insight into its structural aspects as a unit. Changes within 1–3 Å are reasonably acceptable for globular proteins. On the other hand, higher values imply that the protein changes significantly in shape during the simulation.The protein and ligand RMSD values reveal stable binding complexes of carnosine and verapamil, which is used here as a standard P-gp inhibitor . All strUnlike the RMSD, which indicates structural changes in the whole protein, the RMSF calculates the movement of each residue in the protein. Analysis of the protein RMSF values revealed almost similar residue fluctuations in the presence of either carnosine or verapamil . The resThe number of Hbonds formed between P-gp and either carnosine or verapamil was measured during the MD simulation, as shown in The data in To test the ability of carnosine to inhibit P-gp activity, we carried out the rhodamine-123 efflux assay comparing the effects of increasing carnosine concentrations (10–300 μM) to the effect of 10 μM verapamil, a reference standard inhibitor of P-gp. Verapamil treatment of NCI/ADR-RES cells induced the highest intracellular rhodamine-123 accumulation (219.5 ± 35.59%) compared with the control untreated cells . CompareWe evaluated the effect of increasing carnosine concentrations (10–300 μM) on cell viability and doxorubicin-induced toxicity compared with verapamil (10 μM). Interestingly, all carnosine concentrations, like verapamil, significantly reduced cell viability to the same extent  compared with the untreated cells . Figure p < 0.0001). Carnosine at 10–100 μM did not significantly change doxorubicin-induced cytotoxicity. On the other hand, the highest tested concentration of carnosine (300 μM) significantly, but only slightly, potentiated doxorubicin-induced reduction in cell viability . There were no significant differences between the data obtained at different concentrations of carnosine. These data indicate that carnosine-mediated inhibition of cell viability is comparable to that induced by verapamil and that it potentiates doxorubicin cytotoxicity only at higher concentrations (300 μM).Treating the doxorubicin-resistant NCI/ADR-RES cells with doxorubicin (1 μM) for 24 h significantly reduced the viability by 34.6 ± 1.46%. This cytotoxic effect was further increased by concomitant treatment with verapamil  tested in the current work. Another possibility could be that much higher concentrations were required to show a concentration-effect relationship, especially since the anticancer effects observed by others were obtained with carnosine in the millimolar range ,19, unliThe current results revealed only a weak possible potentiation by carnosine of doxorubicin-induced cytotoxicity at 300 µM. The lower concentrations of carnosine (≤100 µM) did not increase the toxicity of doxorubicin. As expected, all cells receiving combined carnosine and doxorubicin treatments were less viable than the corresponding carnosine-only treatments. Although verapamil alone decreased cell viability to levels comparable to carnosine treatments, the combination of verapamil with doxorubicin showed the highest cytotoxic effects. The higher doxorubicin-potentiating effects of verapamil compared with carnosine are in line with our results showing the more potent effect of verapamil in the P-gp inhibition assay and supported by previous findings ,13.The efflux of xenobiotics is a powerful mechanism leading to MDR during cancer chemotherapy ,3. UnforInhibiting the ABC transporters might compromise cellular protection against toxic chemicals; however, it constitutes a great opportunity in the fight against MDR cancer ,7,26. AcThe ligand preparation, docking, and preparation for MD simulation were carried out as previously described, with slight modifications . We seleDesmond, a package from Schrödinger , was used to run MD simulations for 200 ns. The System Builder tool was used to prepare all the systems. Before system creation, the protein was introduced to a DPPC (325 k) membrane. TIP3P was chosen as the Solvent Model having an orthorhombic box . In the simulation, the OPLS 2005 force field was employed. Counter ions were added to the models to make them neutral. NaCl 0.15 M was added to replicate physiological circumstances. For the whole simulation, the NPT ensemble , pressure (P), and temperature (T) are preserved) with 300 K temperature and 1 atm pressure was used. Before the simulation, the models were loosened. The trajectories were stored every 100 ps for examination, and the simulation stability was determined by measuring the peptide RMSD over time. The trajectories were analyzed for RMSF, the radius of gyration (RoG), and protein-ligand Hbonds and distances.excitation = 485 nm; λemission = 535 nm). The mean percentage of rhodamine-123 accumulation was calculated relative to the control untreated cells. The fluorescence emitted by a sample was divided by that of the control, and the product was multiplied by 100.Assessment of P-gp function was carried out in doxorubicin-resistant NCI/ADR-RES cells according to a previously described protocol . Cells w4 cells/well) were cultured for 24 h in DMEM in 96-well plates. Wells were treated with or without carnosine (10–300 μM), verapamil (10 μM), doxorubicin (1 μM), or combinations of doxorubicin (1 μM) with either carnosine (10–300 μM) or verapamil (10 μM). All treatments were maintained for another 24 h, after which 15 μL of MTT  was added to each well. After incubating the cells with MTT for 4 h at 37 °C in the dark, DMSO (100 μL) was added to each well for 10 min to dissolve the formed formazan. A microplate reader was used to measure the absorbance at 540 nm.We assessed the cell viability of doxorubicin-resistant NCI/ADR-RES cells in 96-well plates using the 4,5-dimethyl-2-[3-(3-phenyl-2H-tetrazol-3-ium-5-yl)phenyl]-1,3-thiazole bromide (MTT) assay as previously described . Brieflyp < 0.05.All cell culture data were expressed as the mean percent change from controls ± SD. Data were statistically analyzed using one-way analysis of variance (ANOVA) followed by the Tukey post hoc test for multiple comparisons. GraphPad Prism software version 8.00  was used for the different analyses. Differences between means were significant at P-gp inhibition is a potential target to enhance anticancer drug delivery and efficacy. We have shown by in silico and in vitro approaches that carnosine can inhibit P-gp activity, decrease cell viability, and enhance doxorubicin-induced toxicity at higher concentrations. The docking and MD simulation studies showed that Hbond formation between carnosine and the amino acids GLU875, GLN946, and ALA871 and a stacking interaction with GLU875 are essential to the stability of the carnosine-P-gp complex.


The hydrogels were expected to be able to respond to both temperature and pH changes due to the nature of Alg-g-PNIPAAm and chitosan. The hydrogel formation was determined by a test tube inverting method and confirmed by the rheological measurements. The rheological measurements showed that the PEC hydrogels formed at room temperature could be further enhanced by increasing temperature over the lower critical solution temperature (LCST) of PNIPAAm, because PNIPAAm would change from hydrophilic to hydrophobic upon increasing temperature over its LCST, and the hydrophobic interaction between the PNIPAAm segments may act as additional physical crosslinking. The controlled release properties of the hydrogels were studied by using the organic dye rhodamine B (RB) as a model drug at different pH. The PEC hydrogels could sustain the RB release more efficiently at neutral pH. Both low pH and high pH weakened the PEC hydrogels, and resulted in less sustained release profiles. The release kinetics data were found to fit well to the Krosmyer–Peppas power law model. The analysis of the release kinetic parameters obtained by the modelling indicates that the release of RB from the PEC hydrogels followed mechanisms combining diffusion and dissolution of the hydrogels, but the release was mainly governed by diffusion with less dissolution at pH 7.4 when the PEC hydrogels were stronger and stabler than those at pH 5.0 and 10.0. Therefore, the PEC hydrogels are a kind of smart hydrogels holding great potential for drug delivery applications.Smart hydrogels that can respond to external stimuli such as temperature and pH have attracted tremendous interest for biological and biomedical applications. In this work, we synthesized two alginate- N-isopropylacrylamide) (PNIPAAm) in response to temperature changes makes its copolymers form thermoresponsive hydrogels [Smart hydrogels that can sense and respond to external stimuli, such as temperature, pH, light, and electric fields, have attracted tremendous interest because of their great potential for biological and biomedical applications ,6,7,8,9.ydrogels ,17,18,19ydrogels ,19,20,21ydrogels . The LCSydrogels ,24. Copoydrogels ,26.Alginate (Alg) has been used to copolymerize with PNIPAAm and the copolymers have been reported to form thermoresponsive micelles and hydrogels for drug delivery and other applications ,31,32,33Sodium alginate has been reported to form polyelectrolyte complexes (PECs) with chitosan (Cts) ,40,41,42g-PNIPAAm thermosensitive copolymers  as a model drug. Since both alginate and chitosan are pH-sensitive polymers, the drug release experiments were carried at different pH to study the effect of pH on the release kinetics.In this report, PEC hydrogels were successfully prepared by mixing the Alg-n = 120k–190k Da) was purchased from Sigma-Aldrich. Alginate-g-PNIPAAm copolymers were synthesized as we reported previously [n = 50–190 kDa), rhodamine B, and 2-(N-morpholino)-ethanesulfonic acid (MES) were obtained from Sigma-Aldrich.Sodium alginate , and PMIPAAm contents were synthesized and used in this study  was fixed to be 7.4 wt%. The hydrogels formed by alginate-g-PNIPAAm alone were prepared by dissolving alginate-g-PNIPAAm in water, followed by adding acetic acid solution equivalent to the amount used for dissolving chitosan in the preparation of the chitosan/alginate-g-PNIPAAm PEC hydrogels. The final copolymer contents of the hydrogels were 7.4 wt%. The hydrogel formation was determined by a test tube inverting method. Each sample for hydrogel preparation was carried out in a test tube. The same was considered to have formed hydrogel if it could not flow even when the sample tube was inverted by 180°.The PEC hydrogels formed by alginate-The dynamic rheological measurements were carried out using a HAAKE™ MARS III Rotational Rheometer with parallel plate geometry (35 mm diameter) at a gap of 1.0 mm. Samples were prepared as described in the previous section. The samples were carefully loaded onto the measuring geometry and oil was added around the measuring geometry to minimize the water evaporation during the experiments. The changes in elastic modulus (G′) and viscous modulus (G″) were measured at a fixed frequency of 1.0 Hz and a constant stress of 1.0 Pa with heating from 25 to 37 °C at 0.05 °C/s. Oscillatory stress sweeps were carried out by applying an increasing shear stress logarithmically from 0.1 Pa at a fixed frequency of 1.0 Hz at 37 °C, until the hydrogels were destroyed at a G′/G″ crossover, and a 100% deformation was reached. The yield stress (τ) was defined as the applied shear stress at the G′/G″ crossover .g-PNIPAAm was dissolved in the RB solution. The PEC hydrogels formed by alginate-g-PNIPAAm and chitosan were prepared by mixing the aqueous solution of alginate-g-PNIPAAm with an acetic acid solution of chitosan at room temperature. The weight ratio of alginate segment to chitosan was set to be 17:13.8, which converts to a 1:1 molar ratio of carboxylate group of alginate to amine group of chitosan. The total content of polymers  was 7.4 wt% and the content of RB was 0.1 wt%. The hydrogels formed by alginate-g-PNIPAAm alone were prepared by dissolving alginate-g-PNIPAAm in the RB solution, followed by adding an acetic acid solution equivalent to the amount used for dissolving chitosan in the preparation of the chitosan/alginate-g-PNIPAAm PEC hydrogels. The final copolymer contents of the hydrogels were 7.4 wt% and the content of RB was 0.1 wt%.Rhodamine B (RB) was dissolved in deionized water at 2 mg/mL. Alginate-RB1 is the concentration of RB and A1 is the absorbance value. The calibration curve under the concentration range from 0.00039 to 0.025 mg/mL is linear with a correlation coefficient of R2 = 0.9999.The pre-weighted hydrogels (500 mg) were loaded into a 1 mL syringe. The syringe was incubated at 37 °C for 15 min. Then, the hydrogel was injected into a tea bag and the tea bag was immersed into 30 mL release medium in a centrifuge tube. The sample tube was shaken at 100 rpm at 37 °C in the dark. At predetermined time intervals, 10 mL of release medium was replaced by fresh release medium. The concentration of RB in the release medium was calculated by measuring the absorbance of RB at 555 nm using a microplate reader . RB solutions of various concentrations in PBS buffer were prepared and absorbance at 555 nm was measured to produce a standard curve. From the standard curve the following equation was obtained, where Ct is the total amount of RB released from the hydrogels at time t, and M0 is the amount of RB loaded into the hydrogel.The cumulative drug release was calculated from the following equation:f,p is the amount of polymer remained in the tea bag after release, and M0,p is the initial amount of polymer in the hydrogel.After 100% release of RB from the hydrogels, the tea bags were removed from the release media. The tea bags were weighed after freeze-drying, and the residual polymers of the hydrogels were calculated. The weight percentage of residual polymers in the tea bags after release was calculated from the following equation:RB2 and corresponding absorbance A2 in the acidic solution is below with R2 = 0.9998:The release medium was changed from PBS (pH 7.4) to acetic acid/sodium acetate solution  and sodium tetraborate/sodium hydroxide solution . The linear correlation of RB concentration CRB3 and corresponding absorbance A3 in the basic solution is below with R2 = 0.9999:The linear correlation of RB concentration CtM is the total amount of RB released from the hydrogel at time t, M0 is the amount of RB loaded into the hydrogel, tM/M0 is the fraction of RB released from the hydrogel at time t, K is the release rate constant, and n is the release exponent.The release kinetics and mechanism of RB released from the hydrogels was simulated using a mathematical model well reported in the literature , which in = 0.5), anomalous non-Fickian transport mechanism (0.5 < n < 1.0), non-Fickian Case II relaxational transport , and super case II transport mechanism (n > 1.0).The Krosmyer–Peppas model is an advanced model that includes Fickian diffusion mechanism (1H NMR spectra (3) [2-), while those of NIPAAm monomer between 5.0 and 6.0 ppm disappeared. PNIPAAm with amino end was synthesized by introducing excessive ethylenediamine (EDA) to the end of PNIPAAm-COOH via carbodiimide chemistry. The extra EDA was removed by precipitation in diethyl ether. The clearance of EDA was confirmed by thin layer chromatography with acetone as solvent. The amino end group was quantified by the TNBS assay [2 to alginate was confirmed by 1H NMR spectroscopy , and PNIPAAm contents were synthesized using the method we reported previously . The syn spectra  are listctra (3) , 1.94 ppBS assay . The gratroscopy . Three p31-77% and Alg-PN44-72% copolymers formed solutions at 7.4 wt% in PBS buffer (pH = 7.4), which could flow easily in sample test tube, as we reported previously [31-77% and Alg-PN44-72% at 7.4 wt% when the copolymers were dissolved in acetic acid solutions at room temperature  of the PEC Cts/Alg-PNto 37 °C a,b. Therer 34 °C c,d. The er 34 °C f.g-PNIPAAm and chitosan are much stronger that the hydrogels formed by Alg-g-PNIPAAm alone, apparently because of the strong electrostatic interaction between the negatively charged alginate segments and the positively charged chitosan chains; and 2) the hydrophobic interaction between the PNIPAAm segments at temperatures above its LCST can act as additional physical crosslinking, which largely strengthen the hydrogels (both Alg-PN44-72% and Cts/Alg-PN44-72% hydrogels), making the hydrogels thermosensitive, or “smart” in responding to temperature changes around the LCST of PNIPAAm. The hydrogels formed by Alg-PN44-72% (both Alg-PN44-72% and Cts/Alg-PN44-72% hydrogels) showed clear thermosensitive property but those by Alg-PN31-77% did not, indicating that longer PNIPAAm graft chains are necessary for an Alg-g-PNIPAAm copolymer to form thermosensitive hydrogels under the conditions explored in this study. Similar observations were also reported by us [From the results shown in ed by us ,28 and oed by us ,22,30,55y is the shear stress required to destroy the hydrogel network, which happens at the G′/G″ crossover when the solid-like hydrogel changes into a liquid-like one [y was 5.1 pa for Cts/Alg  in a covered test tube, which was incubated at 37 °C in a water bath shaking at 100 rpm. The rhodamine B (RB) release profiles from the hydrogels are shown in 44-72% > Alg-PN44-72% > Cts/Alg-PN31-77% >> Cts/Alg > Alg-PN31-77% > Alg. Longer PNIPAAm chain in the Alg-g-PNIPAAm copolymer and the formation of PEC could make the hydrogels stronger and stabler, thus reducing the release rate of RB from the hydrogels. So, Alg-PN44-72% exhibited more sustained release than Alg-PN31-77%, and Cts/Alg-PN44-72% and Cts/Alg-PN31-77% more than Alg-PN44-72% and Alg-PN31-77%, respectively.Generally, at pH 7.4 among the six hydrogels or polymers tested for RB release, they provided more sustained release in the order Cts/Alg-PNg-PNIPAAm alone eroded completely after the complete release of RB, whereas 40–70% of the PEC hydrogels remained after the complete release of RB. As a result, the release rates of RB from those non-PEC hydrogels were faster than those corresponding PEC hydrogels. It is thought that the RB release rate depended on the diffusion rate as well as the hydrogel eroding rate. The formation of PEC slowed down the eroding of the hydrogels and resulted in a more sustained release of RB. In a recent report, Cts/Alg PEC hydrogels were additionally crosslinked with divalent cations such as Ca2+ and Zn2+ for enhancing the drug release properties [The hydrogels formed by alginate-operties . our PNIAlginate and chitosan are pH sensitive polymers. The effect of pH on the release of RB from the PEC and non-PEC hydrogels were investigated using acetic buffer (pH 5.0) and borate buffer (pH 10.0). The comparisons of the release profiles for the hydrogels at different pH are shown in 31-77% always gave the fastest release rate due to the short PNIPAAm chains as shown in 31-77% always gave slower release rate than Alg-PN31-77%, and same phenomena were observed for Cts/Alg-PN44-72% and Alg-PN44-72%. Moreover, the longer PNIPAAm chains also slowed down the release rate compared to shorter PNIPAAm chains. Therefore, Alg-PN44-72% gave a slower release than Alg-PN31-77%, and Cts/Alg-PN44-72% exhibited a slower release than Cts/Alg-PN31-77%. Interestingly, both PEC and non-PEC hydrogels formed from Alg-g-PNIPAAm with longer PNIPAAm chains showed less effects of pH changes on their release profiles. This is understandable because PNIPAAm is not a pH-sensitive polymer.Among the non-PEC hydrogels formed by copolymers, the hydrogel Alg-PN31-77% and Alg-PN44-72% hydrogels (31-77% and Cts/Alg-PN44-72% (The acetic buffer (pH 5.0) could reduce the release rate for non-PEC hydrogels formed by copolymers alone, i.e., the Alg-PNydrogels a,c. ProbPN44-72% b,d. In tg-PNIPAAm copolymers. Therefore, the high pH may also weaken the non-PEC hydrogels and then increase the release rate for the non-PEC hydrogels.The borate buffer (pH 10.0) could neutralize the protonated amine groups of chitosan. Therefore, the high pH may weaken the PEC hydrogels and then increase the release rates for the PEC hydrogels. In the meantime, the borate buffer also deprotonate the non-PEC hydrogels formed by the Alg-tM is the total amount of RB released from the hydrogel at time t, M0 is the amount of RB loaded into the hydrogel, tM/M0 is the fraction of RB released from the hydrogel at time t, K is the release rate constant, and n is the release exponent. The value of n is related to the drug release mechanism [There are a number of mathematical models that are used to predict release kinetic models such as the zero order, first order, Higuchi, Korsmeyer–Peppas, and others . In thisechanism .n = 0.5 corresponds to a Fickian diffusion mechanism where the drug release is governed by diffusion, 0.5 < n < 1.0 to anomalous non-Fickian transport, n = 1.0 to non-Fickian Case II  transport where the drug release rate corresponds to zero-order release kinetics and the mechanism driving the drug release is the swelling or relaxation of polymeric chains, and n > 1.0 to super case II transport mechanism [For the case of a planar thin film system, echanism .n and K were obtained by simulating the release profile data in 2) in 2 values of the fittings are all higher than 0.979 while mostly higher than 0.99, indicating that the release kinetics data fitted well to the model. For all four samples with the fitting done in n ranges from 0.521 to 0.709. Taking the structures of the four hydrogel samples into consideration, the release should be governed by a combination of diffusion of RB and dissolution of the hydrogels. The PEC hydrogels Cts/Alg-PN44-72% and Cts/Alg-PN31-77% were stronger and stabler than their Alg-g-PN counterparts, so the release was mainly governed by diffusion with less dissolution. Particularly, at pH 7.4, the values of n for the PEC hydrogels Cts/Alg-PN44-72% and Cts/Alg-PN31-77% are 0.521 and 0.525, respectively, which are very close to n = 0.5. The n values are similar to those of the previously reported stable thermogel release system [n for the PEC hydrogels are higher, ranging from 0.546 to 0.621, indicating the release mechanism contains more dissolution of the hydrogels. For the non-PEC hydrogels Alg-PN44-72% and Alg-PN31-77%, the values of n range from 0.613 to 0.709, indicating that the release was governed by more dissolution than those PEC hydrogel counterparts. The above discussion is in good agreement with the results on the weight percentage of residual polymers after 100% release of RB in (The values of e system . As discof RB in , where tg-PNIPAAm copolymers and the positively charged chitosan in aqueous solutions. It was further proved that the PEC hydrogels were able to respond to both temperature and pH changes due to the natures of Alg-g-PNIPAAm and chitosan. Although the Alg-g-PNIPAAm copolymers could form very weak hydrogels by themselves, much stronger PEC hydrogels could be formed between the Alg-g-PNIPAAm copolymers and chitosan. The rheological study revealed that the strength of the PEC hydrogels was significantly enhanced through the formation of the PECs between the negatively charged alginate segments and the positively charged chitosan polymer chains. In addition, the existence of the PNIPAAm segments in the Alg-g-PNIPAAm copolymers significantly enhanced the strength of the PEC hydrogels upon temperature increasing over the LCST of PNIPAAm, because PNIPAAm would change from hydrophilic to hydrophobic upon increasing temperature over its LCST, and the hydrophobic interaction between the PNIPAAm segments may play a role as additional physical crosslinking. The length of the PNIPAAm in the Alg-g-PNIPAAm copolymers must be long enough to enhance the PEC hydrogels. The PEC hydrogels could sustain the release of the model drug RB from the hydrogels. Low pH may strengthen the non-PEC hydrogels formed by the Alg-g-PNIPAAm copolymers, and then result in more sustained release of RB from the hydrogels. However, low pH may weaken the PEC hydrogels, and resulted in less sustained release profiles. In contrast, high pH may weaken both PEC and non-PEC hydrogels formed by the Alg-g-PNIPAAm copolymers, and then result in less sustained release for the hydrogels. The PEC hydrogels showed more sustained release profiles than the corresponding non-PEC hydrogels. Each hydrogel showed the most sustained release at pH 7.4. Among all hydrogels tested in this study, the PEC Alg-PN44-72% hydrogel showed the most sustained release profiles. The release kinetics data were found to fit well to the Krosmyer–Peppas power law model. The analysis of the release kinetic parameters obtained by the modelling indicates that the release of RB from the PEC hydrogels followed mechanisms combining diffusion and dissolution of the hydrogels, but the release was mainly governed by diffusion with less dissolution at pH 7.4 when the PEC hydrogels were stronger and stabler than those at pH 5.0 and 10.0, as well as those non-PEC counterparts. Therefore, the PEC hydrogels formed by the Alg-g-PNIPAAm copolymers and chitosan are a kind of smart hydrogels that could respond to both temperature and pH changes, holding great potential for drug delivery applications.Our data have demonstrated that the smart hydrogels were successfully prepared through formation of PEC between the negatively charged Alg-


L. reuteri can alter social and stress-related behaviors – yet, the underlying mechanisms remain largely unknown. Although traditional laboratory rodents provide a foundation for examining the role of L. reuteri on the gut-brain axis, they do not naturally display a wide variety of social behaviors. Using the highly-social, monogamous prairie vole (Microtus ochrogaster), we examined the effects of L. reuteri administration on behaviors, neurochemical marker expression, and gut-microbiome composition. Females, but not males, treated with live L. reuteri displayed lower levels of social affiliation compared to those treated with heat-killed L. reuteri. Overall, females displayed a lower level of anxiety-like behaviors than males. Live L. reuteri-treated females had lower expression of corticotrophin releasing factor (CRF) and CRF type-2-receptor in the nucleus accumbens, and lower vasopressin 1a-receptor in the paraventricular nucleus of the hypothalamus (PVN), but increased CRF in the PVN. There were both baseline sex differences and sex-by-treatment differences in gut microbiome composition. Live L. reuteri increased the abundance of several taxa, including Enterobacteriaceae, Lachnospiraceae NK4A136, and Treponema. Interestingly, heat-killed L. reuteri increased abundance of the beneficial taxa Bifidobacteriaceae and Blautia. There were significant correlations between changes in microbiota, brain neurochemical markers, and behaviors. Our data indicate that L. reuteri impacts gut microbiota, gut-brain axis and behaviors in a sex-specific manner in socially-monogamous prairie voles. This demonstrates the utility of the prairie vole model for further examining causal impacts of microbiome on brain and behavior.Research on the role of gut microbiota in behavior has grown dramatically. The probiotic  The vast number of organisms that comprise the gut microbiota have recently become a research area of great focus for human health. Bidirectional connections between the gut and the brain, commonly referred to as the gut-brain axis, allow for an indirect influence of gut microbiota on brain function and resulting behaviors as well as top-down influences on microbial survival . It has L. reuteri (formerly L. reuteri), a common probiotic, has become a focus in some studies. Administration of live, but not heat-killed, L. reuteri upregulates OT in the paraventricular nucleus of the hypothalamus (PVN) and restores prosocial behavior in mice bred from mothers fed high-fat diets  to examine microbial impacts on animal-modeled disorders  and corticotropin releasing factor (CRF), in several animal models . Mice raat diets . Adminispression . These disorders . ExploriMicrotus ochrogaster), a rodent commonly found in grasslands of the United States, is a well-established animal model for examining the neurobiology of social behavior due to their natural behaviors that are easily tested in laboratory settings , and CRF, can be altered by gut microbiota . In the M. ochrogaster) captive-bred at Florida State University. Voles were weaned on postnatal day 21 and housed in Plexiglas cages (20 × 25 × 45 cm) with a same-sex conspecific. Cages contained cedar chip bedding with food and water provided ad libitum. Subjects were kept at 20°C under a 14: 10 h light: dark cycle (lights on at 0700). At the time of testing, subjects had reached adulthood (> 90 days) and were sexually naïve. Behavioral testing was performed after 4 weeks of L. reuteri treatment. Pre- and post-treatment weights and water consumption are shown in Subjects were male and female prairie voles (L. reuteri strain MM4-1A (obtained from the American Type Culture Collection [ATCC] strain PTA-6475) was grown anaerobically in a Gas-Pak system  in MRS medium  at 37°C for 24 h. Under sterile conditions, cells were concentrated by centrifugation, washed and resuspended in phosphate-buffered saline (PBS)/10% glycerol at an optical density at 600 nm (OD600) of 80–100, and immediately frozen in 2 mL aliquots at −80°C. At least 1 frozen aliquot from each live preparation was thawed and plated to MRS agar  to confirm live L. reuteri at a concentration of 4.0E09-1.3E10 colony-forming units (cfu) per aliquot. Aliquots from 2 separate batches of L. reuteri were used to construct 16S rRNA gene V3-V4 amplicon libraries. These were sequenced and analyzed as described below and found to be composed of only L. reuteri sequences  was housed with a same sex cagemate that received the same treatment. Voles from different cages were randomly assigned to receive drinking water with either live L. reuteri or HK (control) L. reuteri. L. reuteri aliquots were administered each morning for 28 days in ddH2O. The dose  used in the current study was adapted from a previous study examining L. reuteri effects on social behavior in mice (We chose to study the difference between HK- in mice . The intvia J-Watcher.The open field (OF) test has been established in our previous study in prairie voles . The opevia J-Watcher. Behaviors quantified included frequency and duration in the conspecific cage, empty cage, and connecting tube. Additional behavioral quantifications included duration and frequency of specific behaviors  test has also been established in our previous study . The tesiors see .via J-Watcher. Behaviors quantified included the duration and frequency in the open arms, closed arms, and the center of the EPM. The percentage of time in the open arms and locomotor activity  were also calculated. During each behavioral test, subjects treated with either live- or HK-L. reuteri were randomly processed.The elevated plus maze (EPM) test has been established and validated in our previous vole study . BrieflyAll subjects were rapidly decapitated immediately after the post-treatment stool collection. Brains were immediately extracted and placed on dry ice. All brains were stored at −80°C until processing. Tissue punches were collected from coronal sections (200 μm thick) from the prefrontal cortex (PFC), amygdala (AMY), nucleus accumbens (NAcc), paraventricular nucleus of the hypothalamus (PVN), and ventral tegmental area (VTA) (four sections per region per subject). Tissue punches were stored at −80°C until subsequent protein extraction.Protein extractions were performed using established methods . BrieflyWestern blotting procedures were performed using our established methods . Brieflyvia J-Watcher and analyzed by two-way ANOVA (SEX X TREATMENT) using IBM SPSS Statistics 19 software . Significant sex-by-treatment interactions were further examined by the Student–Newman–Keuls (SNK) post hoc test. Anxiety-like behaviors for the EPM and OF tests were also analyzed by averaging the z-score conversions for the percentage of time spent in the open arms of the EPM test and duration of time in the center of the OF test. Western blot results were analyzed via one-way ANOVA using SPSS separately for male and female subjects, as different antibodies were used due to discontinuance during experimental processing. The density of Western bands were analyzed by Image Lab software (Bio-Rad), normalized to the reference protein (GAPDH), and presented as the percent change from controls. Due to corrupt video files and typical loss of useable tissue, some subjects were not included in analyses. Significant outliers were removed from analyses. Data are shown as mean ± SEM. Values for each neurochemical marker Western band density relative to the control GAPDH band density are shown in Experimenters were blind to group allocation during behavioral testing and subsequent analyses. All behavioral data were blindly scored L. reuteri treatment, and post-treatment collections occurred in the afternoon on day 2 of behavioral testing. DNA was prepared from −80°C-frozen stool samples using the MoBio/QiaAmp PowerFecal DNA kit, according to manufacturer’s instructions . Isolated DNA was quantified by absorbance at 260 nm on a Nanodrop spectrophotometer and by fluorescence using the dsDNA HS DNA Assay on a Qubit fluorometer .Stool samples were collected using our established procedures . All samLactobacillus/Leuconostoc/Pediococcus-specific 16S rRNA gene primers, F-lacto05 5 ′AGCAGTAGGGAATCTTCCA, R-Lacto04 5′CGCCACTGGTGTTCYTCCATATA (Lactobacillus’); L. reuteri-specific 16S-23S rRNA gene spacer primers, F- L.reuter07 5′CGTACGCACTGGCCCAA; R- L.reuter06 5′TATGCGGTATTAGCATCTGTTTCC  and gene-specific primers at 0.5 μM each in an 8 μL reaction. Each reaction contained 3.3 ng bacterial DNA as determined by Qubit. Cycling conditions were: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 53°C for 30 s and 68°C for 30 s. 60–95°C melt curve analysis following PCR was performed using default settings. Gene-specific primers were: 16S rRNA gene V3-V4 variable region amplicon primers (V3-V4) .Raw paired-end 16S rRNA gene sequences had their first 5 base pairs trimmed and were truncated to a length of 280 based on quality score summaries visualized with Multi-QC . Sequenc® Ultra™ II DNA Library Prep Kit for Illumina , following the manufacturer’s protocol. 500 ng total DNA was found to be sufficient for high-yield libraries. For the 32 samples from the 16 L. reuteri-treated females, the average library fragment size was 885 bp. Sequencing library and library pools quantified by KAPA assay. The shotgun metagenome sequencing of 32 sample libraries from the 16 L. reuteri-treated females was performed on an Illumina HiSeq 2500 sequencer in the Florida State College of Medicine Translational Science Laboratory. Paired-end 250-base sequence reads were generated in the cBOT Rapid Run protocol. The Metagenome-ATLAS  pipeline was used for whole-genome shotgun sequence assembly, annotation and genomic binning  on the DESeq2-normalized count tables was also performed using Microsoft Excel. Briefly, in this method, differences in representation of each microbial taxon were determined for each individual animal pre- to post-L. reuteri treatment. For both methods, a filtering criteria of an average log2 fold change of ≥|0.693| was used to select taxa of interest. The VBV pairwise method takes into account the individual variability of animals by requiring that each taxon have a supermajority of animals in the group meet this log2fc criterion. In addition, the method requires that the average log2 fold change of all animals in the group be ≥|1.25| in the same direction. Supermajority cutoffs were 5/8 for each sex-specific probiotic treatment group.The feature count table was normalized using the DESeq2  R packagMean relative abundance of taxa in the different sample groups was calculated using the phyloseq R package . First, p value ≤0.05 in two-tailed Mann–Whitney U tests for two independent samples. Significant differences for pre- vs. post-treatment were defined as those with a p value ≤0.05 in two-tailed Wilcoxon matched-pairs signed rank tests.Both the alpha and beta diversity metric analysis were performed using the phyloseq R package  suppleme2 values to determine goodness of fit, and an F-test to determine significance (myplotbetadisp.r – Pastebin.com). The above-mentioned sequencing and analyses of stool samples were performed according to the same protocol as in our recent study in prairie voles  = 10.51, p < 0.01].As y weight . There wF = 4.12, p < 0.05]. There were no sex differences in the frequency of open arm entries  or in overall locomotor activity . No significant treatment effects nor sex-by-treatment interactions were found in the EPM test. In the OF test  = 7.39, p < 0.05] and entered the center more frequently  in comparison to males. There were no sex differences in the number of total line crosses . No other significant effects of treatment nor sex-by-treatment effects were found in the OF test.Our data indicated overall sex differences in anxiety-like behavior in both the EPM and OF tests. In the EPM test , female  OF test , femalesF = 7.42 p < 0.01]. Taken together, these data indicate that, compared to males, females consistently displayed lower levels of anxiety-like behavior.When converting these data into z-scores to compare across behavioral assays, we replicated our effect, where females had a significantly lower average anxiety composite z-score in comparison to males , where live-treated females spent significantly less time interacting in comparison to HK-treated females. This phenomenon was not found in male voles  = 0.46, p = 0.50], treatment differences , nor sex-by-treatment interactions  in the total frequency of social interactions with the conspecific  = 39.70, p < 0.01] and frequency  of anxiety-like behavior in comparison to male voles  = 5.55, p < 0.05] and CRFR2  in the NAcc and V1aR in the PVN , but higher CRF expression in the PVN , compared to HK-treated females. In males, live-treated voles had greater CRF expression in the AMY in comparison to HK-treated ones . No other significant treatment differences were found in measured neurochemical markers. Values for each neurochemical marker Western band density relative to the control GAPDH band density are shown in A variety of neurochemical markers in selected brain areas known to be involved in anxiety-like and social behavior were assayed by Western blot and quantified . Our datL. reuteri relative to the 16S rRNA gene V3-V4 amplicon (ΔΔCt)  .  . The pre-treatment alpha diversity was also greater for females at the family level measured by the Shannon ENS diversity metric . There were no significant differences in the Inverse Simpson’s diversity metric, which weights taxa evenness over richness  . (See Su= 0.40). . Prior t= 0.40).  between = 0.40).  in the qntestine . There w reuteri . The alpare taxa  at the f = 0.58) , and at y metric , which wL. reuteri administration on microbiome composition in male and female prairie voles. Due to the high level of individual variation between animals, the fold change pre- to post-treatment was determined for each individual animal and the average fold change for the group was calculated from these individual fold changes. Inoculation with live L. reuteri for 28 days produced a mean 93.2-fold increase in males (L. reuteri DNA detected by qPCR (relative to 16S rRNA gene V3-V4 amplicon) and a 7.9-fold increase in females. The difference in fold change between the sexes was highly significant . We also found that the relative quantities of L. reuteri in post- vs. pre- live-treated animals were significantly higher for both males  and females . Treatment with HK L. reuteri produced a very small 1.3-fold increase in L. reuteri in both males and females with no difference between the sexes . The difference between pre- vs. post-HK treatment groups was not significant for either sex  and females , but the differences for pre-treatment groups were not significant for either sex . There were no significant differences in the L. gasseri–L. johnsonii species group between the treatment groups  between sample groups, there were no significant differences in alpha diversity by Chao1, Shannon ENS, or Inverse Simpson’s in the live- vs. HK-treated or pre- vs. post-treatment groups. This is in agreement with the 16S rRNA gene data alpha diversity for females . Additional differences in composition between live- vs. HK treatment groups were only for 16S rRNA gene analysis and only at the family level. Live- vs. HK-treated males differed significantly by Bray-Curtis and Weighted UniFrac  on a ‘Vole-by-Vole’ (VBV) pairwise basis for each individual animal prior to comparison of the sample groups (see Methods).In determinations of beta-diversity, if one group has very little variation among the samples that make up the group (a low level of dispersion) and the other group has very large variation (a high level of dispersion), this can have an impact on the significance of the beta-diversity metrics, although PERMANOVA is less sensitive to sample dispersion differences than other analysis methods . We testBacteroidetes  or the family Prevotellaceae  and the Lachnospiraceae  than by 16S rRNA gene V3-V4, despite the fact that these analyses were conducted on the same DNA samples. The most abundant strain was classified by QIIME2 in 16S rRNA gene analysis as a Muribaculaceae (S24-7) uncultured bacterium , with most of this split between the Ruminococcaceae (2–4% of bacteria) and the Lachnospiraceae (2–6%). Since the 16S V3-V4 analysis and the WGS analysis were done on the same DNA samples, the results suggest bias is introduced by one or both of these methods. The more likely possibility is that the V3-V4 amplicon primers have a bias against detection of one or more Bacteroidetes strains in our samples. The MAGs extracted from our WGS vole data have poor recovery of 16S rRNA genes .Measured by the 16S rRNA gene V3-V4 amplicon, the average most abundant phylum in vole stool was ae 4–8%; . Next inae 4–8%;  with mos 11–18%; . These d 11–18%; . As in troidetes , but the 36–42%, , wherease family , and thianalysis . FirmicuNA genes , so it hNA genes . DespiteNA genes . Howeverp < 0.1 adjusted for multiple comparisons) Ruminococcus 1 uncultured rumen bacterium that increased post-treatment. In live-treated males, a different Ruminococcus 1 species decreased, and a Desulfovibrio genus bacterium increased. In HK-treated females, another species of the Ruminococcaceae family and an [Eubacterium] coprostanoligenes group species increased, whereas a Roseburia species decreased. In HK-treated males an Ileibacterium uncultured bacterium increased and a Ruminococcaceae UCG-014 uncultured bacterium decreased. At the family level, by the whole group method, only 3 bacterial families had significant changes and all of these were in lower-abundance lineages  pairwise basis (see Methods). Taxa that had an individual log2fc of |0.693| in the same direction in at least 5 out of 8 animals and had an average log2fc of |1.25| for all 8 animals are shown in 2fc in each animal for each species is shown in Lachnospiraceae NK4A136 group, a species from the Ruminococcaceae family, and an uncultured rumen bacterium from the Gastranaerophilales , and the Bacteroidales p-251-o5. Decreasing in the live-treated males were species from the Elusimicrobium and the Intestinimonas genera, in addition to the aforementioned Gastranaerophilales uncultured rumen bacterium. All of the family-level differences in live-treated animals were in groups also detected at the species level. In HK-treated females there were increases in a Bacteroidia family, the Helicobacteriaceae, and a family of Gastranaerophilalaes. In HK-treated males, the Anaeroplasmataceae and Erysipelotrichaceae families increased, whereas the Christensenellaceae family decreased.In this study and our previous work , we obsephilales . Live-trncreased . This sap method . A Rumined males , and by ed males . Also inL. reuteri treatment changes produced post-treatment differences in some of these taxa between live- vs. HK-treated groups. Of 11 species that were differentially abundant between live-treated and HK-treated females  group. Decreasing in live-treated females was the xylan-degrading species Dysgonomonas sp. BGC7  butyrate are increased in the HK-treated females than in the live-treated  negatively correlated with CRFR2 in the NAcc in females. In males, this same species positively correlated with time in the open arms of the EPM and increased post-treatment in 5/8 HK-treated males  also negatively correlated with CRFR2 in the NAcc and its post-treatment abundance positively correlated with SA corner time , increases in two species,  the PVN . Bifidob females  and decr females . The BifF center , and it F center . The stralactiae . S. galatermined . At the the NAcc . There weriaceae . The Ach females . CRF in  overall . One of  females .Methanobrevibacter oralis and the strongest negative correlation was with Thermophagus xiamenensis  vs. restoration  is intriguing, as sex-specific neurochemical regulation of social behaviors have been reported in prairie voles  axis, and this increased HPA activity has been previously shown to impair social contact/affiliation in female prairie voles  in several taxa of gut microbiota. The most prominent increasing taxon in this group is Clostridiales bacterium VE202-01. This strain is part of a consortium of Clostridia being developed as a therapeutic for inflammatory bowel disease  was also strongly correlated with social affiliation in cohoused prairie voles  responsible for the effects we observed must also be L. reuteri cell-associated. Cell components responsible for these effects could be polysaccharide, protein, lipid or heat-stable metabolites made more accessible to other members of the gut community by prior heat killing. Material derived from the HK cells could also interact directly with the host gut epithelium as an innate immune or metabolic signal  can be found in the article/All procedures were approved by the Institutional Animal Care and Use Committee at Florida State University and were in accordance with the guidelines set forth by the National Institutes of Health.MD conceived the study. MD, CM, GP, JC, KJ, and ZW designed the study. MD, CM, GP, AB, BW, DT, YL, and KJ performed the experiments. MD, CM, ML, GP, AB, BW, TC, KJ, and ZW analyzed the data. MD, CM, KJ, and ZW wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Institutes of Health  to ZW; partially supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, award number 2014-67013-21579 to KJ; and MD was supported by the NIH program training grant . For MD writing of manuscript was supported by the VA Office of Academic Affiliations, Advanced Fellowship Program in Mental Illness Research and Treatment, Department of Veterans Affairs.ML and TC were employed by Metagenom Bio Life Science Inc.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aimed to analyze the pathogenic characteristics of community-acquired pneumonia (CAP) in a children’s hospital before and after the coronavirus disease 2019 (COVID-19) pandemic and to provide testimony for preventing CAP in the future.A retrospective analysis was performed. The information was collected from the electronic medical record system of the hospital. A total of 2739 children were included from February 1, 2019, to January 31, 2021.Z = − 7.885, P < 0.001). After the pandemic, the proportion of CAP in school-age children and Mycoplasma pneumoniae pneumonia (MPP) and influenza virus pneumonia (IVP) decreased significantly. During the pre-COVID-19 period, the proportions of detected pathogens were as follows: MP (59.56%) > bacteria (50.42%) > viruses (29.57%) > fungi (3.43%). During the post-COVID-19 period, the pathogen proportions were bacteria (56.53%) > viruses (53.60%) > MP (23.47%) > fungi (3.73%).Among these 2739 patients were 1507 (55.02%) males and 1232 (44.98%) females; the median age was 3.84 years. There were 2364 cases during the pre-COVID-19 period and 375 cases during the post-COVID-19 period. The number of hospitalized children after the pandemic was 84.14% lower. The median age after the onset was 1.5 years younger than that before the onset (4.08 years old) (There was a significant decrease in the number of children with CAP hospitalized after the pandemic, especially among school-age children, and the pathogen proportions of CAP with MP and IV were significantly decreased. We inferred that CAP was effectively prevented in school-age children because of the strong mitigation measures. Community-acquired pneumonia (CAP) is the leading cause of morbidity among childhood respiratory diseases. The World Health Organization (WHO) estimates that nearly one-fifth of childhood deaths are caused by pneumonia, which claims more than 2 million lives annually . The gloA retrospective analysis was used in our study, which was conducted in a 400-bed children’s hospital in Beijing, China from February 1, 2019 to January 31, 2021. Children between ≥ 28 days and 18 years of age who completed pathogen testing within 48 hours after admission and met the diagnostic criteria for CAP were included in this study. Referring to “Zhu Futang’s Practice of Pediatrics” (8th edition), our diagnostic criteria  DNA or RNA results indicated a viral infection, and others were diagnosed by clinicians after comprehensive consideration; (3) community-acquired An electronic medical record system called EMR developed by the software company “Beijing Jiahe Meikang Information Technology Co., Ltd.” was used to collect medical records. The specific steps were as follows: (1) “pneumonia” was input in “diagnosis”; (2) “2019.02.01–2022.01.31” was input in “time”, combined with the inclusion and exclusion criteria to select the cases that met the requirements for the study.U test was used to assess the statistical significance of groups. Categorical variables were expressed as frequencies and percentages. Chi-square and continuity correction of Chi-square tests were used to compare groups.Statistical procedures were carried out using SPSS version 26.0. Measurement data with a non-normal distribution were expressed as medians and quartiles. The Mann–Whitney P > 0.05). The median age after the outbreak (2.58 years) was 1.5 years younger than the median age before the outbreak (4.08 years), and the difference was statistically significant . The proportion of CAP in the infant group increased, and in the school-age group, it decreased significantly (P < 0.001). The positivity rate for pathogen detection decreased in spring and summer and increased in autumn and winter during the post-COVID-19 period (P < 0.05). A total of 650 patients with chronic diseases were included in this study, including bronchial asthma, bronchiectasis, tracheal malformation, esophageal dilatation, congenital heart diseases, hypertension, epilepsy, nephrotic syndrome, leukemia, hyperthyroidism, allergic purpura, congenital genetic metabolic diseases, immune deficiency and other chronic diseases of various systems. The number of CAP patients with chronic diseases during the post-COVID-19 period was significantly greater (P < 0.001)  males and 1232 (44.98%) females for a male to female ratio of 1.2:1. The median age was 3.84 (interquartile range: 1.78–6.98) years (ranging from 29 days to 18 years of age). There were 2364 cases during the pre-COVID-19 period and 375 cases during the post-COVID-19 period. The number of hospitalized children after the pandemic was 84.14% lower. No significant difference in sex distribution was observed (P < 0.001). Gram-positive bacteria were the main bacteria. The top three bacteria before and after the pandemic were Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus. In addition, the rates of Pseudomonas aeruginosa and Acinetobacter baumannii also increased after the outbreak.Among the 2739 children with CAP, a total of 2384 pathogenic tests were positive, with 2079 before the pandemic and 305 after the pandemic. The number of CAP cases in children declined substantially post-COVID-19 (Table P < 0.001). All patients after the pandemic in this study tested negative for SARS-CoV-2.There were no significant changes in RSV proportions, and RSV still ranked first. The adenovirus (ADV), influenza virus (IV), and parainfluenza virus (PIV) proportions all decreased after the pre-COVID-19 period, especially IV infection, which nearly became zero. The proportions differed significantly (P < 0.001)  Table .Table 3TThe number of children hospitalized with CAP during the post-COVID-19 period was 84.14% lower than the number hospitalized during the pre-COVID-19 period, especially among school-age children. We inferred that the decline after the outbreak was attributed to the strict prevention and control measures implemented in Beijing, such as hand hygiene practices, wearing surgical masks when in crowded places, maintaining a social distance of more than one meter, measuring body temperature and discouraging students with respiratory symptoms from going to school. All of the above factors protected children from infection, especially school-age children.The reason for the decline in CAP in children in the spring and summer after the pandemic is that the pandemic situation was severe at that time. Beijing launched a high-level emergency response and implemented various measures, such as encouraging home isolation, measuring body temperature, checking access cards, scanning health codes and trip codes, and so on to reduce personal activities and the spread of the disease.In addition, our study showed that the age of children suffering from CAP decreased by 1.5 years after the onset of the pandemic. We believe that this was because the proportion of CAP in school-age children decreased significantly, but the proportion in infant and younger children did not. For the same reason, the proportion of CAP patients without chronic disease decreased significantly, but the proportion of CAP patients with chronic disease did not. Infants, younger children and children with chronic diseases had hypoimmunity. Despite the above protective measures, these children were still more susceptible to infection than school-age children and children without chronic diseases. Another reason for the higher proportion of children with chronic diseases was that they tended to be sicker after infection, and parents were more motivated to seek medical attention.In our study, a pathogen was detected in 2079 of the 2364 children (87.94%) during the pre-COVID-19 period and in 305 of the 375 children (81.33%) during the post-COVID-19 period, which differed from some other studies [In our study the positivity rate of MP decreased from 59.56% to 23.47%; furthermore, the positivity rates of ADV, IV, and PIV all decreased during the post-COVID-19 period, especially influenza virus pneumonia, which was nearly zero. Many reports before the outbreak showed that MP was the most common pathogen of CAP in children. Atkinson et al. , 11. TheStreptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus, which was in accordance with the results of Nathan et al. [Klebsiella pneumoniae was the most common bacteria. The reasons may be related to the region, time and climate. The increased proportions of P. aeruginosa- and A. baumannii-associated CAP after the outbreak may be related to the increased proportion of CAP patients with chronic disease.The experiment also showed that Gram-positive bacteria were the main strain detected before and after pandemic onset, and the top three bacteria were n et al.  and Mathn et al.  but was n et al.  and Liu n et al. , who repThis study showed that the positivity rate of coinfections was higher than that of any single pathogen before and after the onset of the pandemic, which was much higher than the 29% reported in the study by Juvén et al. . The reaA limitation of this study is that it included only a single pediatric center. We look forward to more relevant reports to provide a basis for developing countries to formulate pediatric CAP prevention and control measures.In conclusion, there was a significant decrease in the number of children hospitalized with CAP after the pandemic, especially among school-age children, and the pathogenic proportions of CAP with MP and IV were significantly decreased. We inferred that CAP was effectively prevented in school-age children because of the strong mitigation measures. This may help health managers, medical workers and educators develop policies and measures to prevent CAP in the future.


Selenium nanoparticles (SeNPs) are among the emerging selenium supplements because of their high bioactivity and low toxicity. However, bare SeNPs are prone to activity loss caused by aggregation and sedimentation. This study aims to stabilize SeNPs with curdlan (CUR), a polysaccharide, to maintain or even enhance their biological activity. Herein, the stable SeNPs were constructed via the unique conformational transition of CUR induced by alkali-neutralization (AN) pretreatment. The physicochemical properties and structures of the prepared SeNPs were characterized by dynamic light scattering (DLS), UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and free-radical-scavenging activity assays. The results show that most SeNPs are stabilized within the triple helix of CUR that has been pretreated with high-intensity AN treatment. These amorphous, small-sized (average size was 53.6 ± 17.7 nm), and stabilized SeNPs have significantly enhanced free-radical-scavenging ability compared to the control and can be well-stabilized for at least 240 days at 4 °C. This work indicates that CUR, as a food additive, can be used to well-stabilize SeNPs by AN pretreatment and provides a facile method to prepare and enhance the stability and bioactivity of SeNPs via triple-helix conformational transition. The reduction in selenite with ascorbic acid can be described by the general reaction as follows [j = 0–2 and i = 0–2. However, SeNPs are prone to aggregate into large particles without stabilizers, which can seriously affect their bioavailability and bioactivity [Selenium plays a key role in regulating immunity and sperm activity , and sel2SeO3)) ,5,6,7. S2SeO3))  or by us2SeO3))  or can b2SeO3)) . The maiosulfate ,12,13,14 follows .HjSeO3jactivity . TherefoAuricularia polysaccharide and lentinan [Natural polysaccharides are increasingly being used as stabilizers for SeNPs because of their excellent biocompatibility and the large number of hydroxyl groups . Chitosalentinan ,29. Howelentinan , which mβ- glucan without branched chains [Curdlan (CUR) is a linear d chains ,32, whicd chains . CUR is d chains , the safd chains . In brieCNaOH/HCl = 0.1 mol/L), the THAs first disaggregate into small-sized THAs and independent triple helices (ITHs) after alkali treatment; then, these ITHs re-aggregate to THAs after the neutralization treatment. Under a high-intensity AN treatment (CNaOH/HCl = 1.0 mol/L), THAs first disaggregate completely into random coils after alkali treatment; then, these random coils renature to ITHs, but few ITHs further aggregate to THAs. The difference between the two self-assembly behaviors of CUR lies in the presence or absence of unwinding-rewinding of ITHs. From this, it can be hypothesized that different self-assembly behaviors of CUR may affect the structure and physicochemical properties of CUR-SeNP composites because the SeNPs may be encapsulated inside the triple helix through the renaturation of ITHs. Therefore, there is a need to verify how this difference significantly affects (if there is any effect) the physicochemical properties of CUR-SeNP composites.According to our previous confirmation, CUR has different self-assembly behaviors at different AN concentrations . Under aThis study aims to stabilize SeNPs using unmodified CUR and to resolve the effects of CUR with different intensities of AN pretreatment on the stabilization of SeNPs. We found that high-intensity AN-pretreated CUR was more suitable for stabilizing SeNPs, which have good storage stability and antioxidant activity. This work would help to promote the use of CUR, a food additive, in the fabrication of selenium supplements that are of interest in the field of food nutrition and functional foods.w = 1.55 × 104 Da) was purchased from Yuanye Bio-Technology Co., Ltd.  and used without any additional purification [w cutoff = 3500 Da) was purchased from Solarbio Science & Technology Co., Ltd. . Sodium selenite (Na2SeO3) and ascorbic acid were purchased from Sigma–Aldrich Chemical Co., Ltd. . All chemicals and solvents were of analytical grade, and ultrapure water was used in all experiments.Curdlan  pretreatment was performed according to our previous work with slight modifications . In brie2SeO3 aqueous solution, and magnetically stirred for 1 min. Then, 500 μL of 0.2 mol/L ascorbic acid aqueous solution was separately added into the mixtures to start the synthesis reaction of SeNPs, and the resulting mixtures were stirred for 24 h in the dark at 25 °C. At the end of the reaction, the mixtures were dialyzed against ultrapure water for 48 h, and the water was changed every 12 h. After the necessary analysis of the dialyzed sample, it was freeze dried with a SCIENTZ 18N  freeze dryer and stored at 4 °C and 25 °C. The samples were named CUR-SeNPs-L and CUR-SeNPs-H according to the intensity of AN pretreatment. Control sample without CUR was prepared as above and named SeNP control. Control samples without SeNPs were prepared as described previously and named CUR-L and CUR-H according to the intensity of AN pretreatment.CUR-SeNPs were prepared according to the following procedure. The reaction of sodium selenite with ascorbic acid was carried out as described by Li, Li, Wong, Chen, Zhang, Liu, and Zheng  with sliThe hydrodynamic-diameter distribution and zeta potential of the CUR-SeNP composites were measured by dynamic light scattering (DLS) with a ZETASIZER NANO ZS90  at 25 °C . The dilAbout 2 mL of the diluted sample was drawn into a quartz cuvette and transferred into a corrected UV-2501(PC)S  UV-vis absorption spectrometer. The scanning wavelength range was 200–800 nm with a resolution of 1 nm .−1 and at a resolution of 0.5 cm−1. Each sample was scanned 16 times. The results were analyzed by an OPUS 8.5 .The KBr-disk method was used for FTIR analysis . BrieflyThe lyophilized sample was suitably ground to powder and measured by a D/MAX 2500 V  X-ray diffractometer using Bragg–Brentano  geometry and Cu-Kα radiation. The operational parameters were as follows: scanning range of 5–80°; scanning speed of 4°/min; and the voltage and current were 40 kV and 40 mV, respectively . The resThe samples were observed with a HT-7700  transmission electron microscope . In a ty4 aqueous solution, 1.0 mL of 9.0 mmol/L salicylic acid ethanol solution, and 1.0 mL of sample were mixed. After adding 1.0 mL of 9.0 mmol/L H2O2, the mixture was incubated for 30 min at 37 °C. The absorbance was measured at 510 nm. The hydroxyl-radical-scavenging ability was calculated using the following equation:1, A2, and A0 represent the absorbance in the presence of sample, the absorbance in the absence of H2O2 (water instead), and the absorbance in the absence of sample (water instead), respectively.Hydroxyl-radical-scavenging activity was determined by the procedure described by Mao, Zou, Feng, Wang, Zhao, Ye, Zhu, Wu, Yang, and Wu  with sli2EDTA. The absorbance was measured at 325 nm every 30 s for 10 min. The superoxide-radical-scavenging ability was calculated using the following equation:0 represents the increment in A325nm of the mixture without a sample, and ΔA1 represents the increment in A325nm of the mixture with a sample.Superoxide-radical-scavenging activity was determined by the procedure described by Li  with slip < 0.05.All determinations were performed in duplicate, and the results are presented as the mean ± standard deviation. Statistical analysis was conducted using IBM SPSS 20.0 software. An analysis of variance (ANOVA) using Turkey’s multiple-range test was performed for mean comparison at max) of the three samples were 497, 258, and 264 nm, respectively, and their absorbances started to increase significantly from 600 nm, which was consistent with the typical UV-vis spectroscopic pattern of SeNPs [max (266 nm), although they are very different in conformation [max. For example, the λmax of SeNPs with an average particle size of 70.9 ± 9.1 nm is approximately 270 nm, while the λmax of SeNPs with an average particle size of 101.6 ± 9.8 nm is approximately 300 nm. Based on this relationship, it is reasonable to assume that the λmax of SeNPs in both CUR-SeNPs-L and CUR-SeNPs-H should be less than 266 nm, and the λmax of the former is smaller. Therefore, the larger SeNPs in CUR-SeNPs-H resulted in a weaker blueshift (266 nm to 264 nm) in λmax, while CUR-SeNPs-L had a stronger blueshift (266 nm to 258 nm).Generally, at a given selenium concentration, the SeNPs with smaller particle size exhibit a more yellowish and more transparent appearance ,24,36. Aof SeNPs . The intormation . Howeverormation , the SeNDh) distributions of the samples were measured, and the results are shown in Dh. As shown in Dh distribution by intensity and volume demonstrated this pattern with increased clarity. The P3 peaks with an average Dh of 4737 nm and 4978 nm (by intensity) in CUR-SeNPs-L and CUR-L confirmed the presence of THAs in the solution. The size of THAs was reported to be in the micrometer scale [Dh distribution, it is reasonable that the P2 peak was attributed by independent triple helices (ITHs) dissociated from THAs. Furthermore, as shown in Dh-P2 (both by intensity and volume) of both CUR-SeNP samples was higher than that of their control samples without SeNPs, indicating that SeNPs were bound to ITHs. However, the binding between SeNPs and CUR was different in the two CUR-SeNP samples. As we described in the introduction, CUR has different conformational transitions induced by different intensities of AN pretreatment. In the formation of CUR-SeNPs-L, SeNPs cannot be wrapped inside the ITHs through the reaggregation of ITHs to THAs, so SeNPs can only be attached to the outside of the ITHs or be trapped inside the re-aggregated THAs [Dh-P2 (both by intensity and volume) of CUR-SeNPs-H compared to CUR-SeNPs-L.To further define the particle size of CUR-SeNP samples, the hydrodynamic diameter  of SeNP control, CUR-SeNPs-L, and CUR-SeNPs-H were measured at −25.3, −20.7, and −15.8 mV, respectively . The zet−1 (stretching vibration of –OH), 2881 cm−1 (stretching vibration of C–H), 1606 cm−1 (absorption peak of the adsorbed water blending vibration), 1002 cm−1 (stretching vibration of C–O), and 890 cm−1 (β-glycosidic linkage) [FTIR and XRD are effective methods for analyzing the structural characteristics of CUR-SeNPs. linkage) . The dif−1 in all spectra, indicating the presence of curdlan in all samples. The –OH stretching vibration peaks of both CUR-SeNP samples were shifted to the lower wavenumbers (3379 cm−1 and 3376 cm−1) compared to that of AN-pretreated CURs (3385 cm−1), indicating that SeNPs were stabilized by the interaction between Se atoms and hydroxyl groups, and this result was similar to that of the other THP-SeNP composites [−1 was attributed to the adsorbed water blending vibration; the slightly lower wavenumber of CUR-H indicated its higher water solubility, and the addition of SeNPs had no obvious effect on this peak.There is a characteristic absorption peak of the β-conformation at 890 cmmposites ,24. Frommposites . Therefomposites , but the−1 in CUR-SeNPs-L and 3376 cm−1 in CUR-SeNPs-H) indicated the different structures of the CUR-SeNPs prepared at different AN pretreatment intensities.Overall, the FTIR results showed that the SeNPs were stabilized through the hydrogen bonding interactions, and the different positions of broad peaks in the spectra of the two CUR-SeNPs samples . Song, Chen, Zhao, Sun, Che, and Leng  suggeste6 primary hydroxyl groups converted to carboxylate groups in carboxymethyl chitosan molecules compared to chitosan, which may reduce the total number of hydroxyl groups [The reactivity of CUR-SeNPs with hydroxyl radicals is shown in l groups . In our 50) was approximately 6.58 μg/mL (selenium concentration). The reactivity of CUR-SeNPs with superoxide anion radicals is shown in Furthermore, the concentration-scavenging ability of all samples showed a concentration-dependent increase, and the corresponding half-inhibitory concentration , large-sized THAs first disaggregated to small-sized THAs and some ITHs under alkali treatment. After neutralization treatment and initiation of the synthetic reactions of SeNPs, some SeNPs were trapped inside the THAs through the re-aggregation of ITHs while the remaining SeNPs failed to be stabilized and thus dispersed in water. When the intensity of AN pretreatment was high (CNaOH/HCl = 1.0 mol/L), THAs first disaggregated and denatured to random coils under alkali treatment. After neutralization and initiation of the synthetic reactions of SeNPs, most SeNPs were stabilized and encapsulated inside the triple helices through the rewinding of ITHs, whereas a few SeNPs were stabilized on the outside of the triple helices or dispersed in water. Obviously, the AN pretreatment intensity determined whether or not SeNPs can be effectively encapsulated inside the triple helix, which was crucial to the stability of SeNPs.Based on all characterization results, the construction mechanisms of CUR-SeNPs under different AN pretreatments were derived. As shown in CNaOH/HCl = 0.1 mol/L), only the disaggregations/aggregations of THAs were present, so most SeNPs were free in water and a few SeNPs were trapped in THAs. At a high AN pretreatment intensity (CNaOH/HCl = 1.0 mol/L), the ITHs dissociated from THAs undergo further unwinding-rewinding, so most SeNPs were encapsulated inside the cavity during the rewinding of ITHs. From the latter route, SeNPs were better stabilized and dispersed by Se···H hydrogen bonding interactions and physical wrapping of a triple helix, and the CUR-SeNPs had a better antioxidant activity.All results confirmed the hypothesis that different AN pretreatments induced different self-assembly behaviors of CUR, which in turn resulted in prepared CUR-SeNPs with various structures, stabilities, and dispersion properties. At a low AN pretreatment intensity The underlying mechanism of the Se···H hydrogen bonding interaction between SeNPs and CUR (or other polysaccharides);(2)The maximum loading of SeNPs in CUR helices and methods for their detection;(3)Further investigation of other biological activities of CUR-SeNPs, such as anti-tumor activity, immunomodulatory activity, etc.In the future, further research and analysis are needed in the following areas, some of which we have already undertaken:


Polyphenol-rich foods are gaining popularity due to their potential beneficial effects in the prevention and treatment of cancer. Foxtail millet is one of the important functional foods, riches in a variety of biologically active substance. Our previous study showed that ferulic acid (FA) and p-coumaric acid (p-CA) are the main anticancer components of foxtail millet bran, and the two have a significant synergistic effect. In the present study, the clinical application potential of FA and p-CA (FA + p-CA) were evaluated in vivo and in vitro. The FA and p-CA target gene enrichment analysis discovered that FA + p-CA were associated with aerobic glycolysis. It was further shown that FA + p-CA remodel aerobic glycolysis by inhibiting the glycolysis-associated lncRNA 495810 and the glycolytic rate-limiting enzyme M2 type pyruvate kinase (PKM2). Moreover, PKM2 expression was positively correlated with lncRNA 495810. More interestingly, the exogenous expression of lncRNA 495810 eliminated the inhibitory effects of FA + p-CA on aerobic glycolysis. Collectively, FA + p-CA obstruct the aerobic glycolysis of colorectal cancer cells via the lncRNA 495810/PKM2 axis, which provides a nutrition intervention and treatment candidate for colorectal cancer. Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system, with high morbidity and mortality ,2. In reColorectal cancer cells exhibit altered energy metabolism, particularly abnormal activation of glycolysis pathways ,5. CompaPolyphenols, ubiquitous in plant-derived cereals, fruits, and vegetables, have been shown to prevent CRC by modulating glycolysis. Accumulating evidence suggests that resveratrol , chrysinIn recent years, the active ingredients in functional foods have received increasing attention as a rich source of potential anticancer drugs ,12. MillTo detect the inhibitory effects of FA + p-CA on CRC cells, p-CA and FA were artificially mixed at a ratio of 1:1.13 . Followiwww.metascape.org) for gene enrichment analysis via WikiPathways. The results indicate that the FA + p-CA target genes were enriched in the aerobic glycolysis pathway  attracted our attention due to it being the most downregulated one upon FA + p-CA treatment, which may be involved in glycolysis. To identify the consequence of RNA-Seq, the expression of lncRNA 495810 in AOM/DSS-induced CRC mice and different colon cancer cells was detected after exposure to FA + p-CA. The results indicated that the expression of lncRNA 495810 in the AOM/DSS group was increased, whereas additional treatment of FA + p-CA decreased its expression C. CorresFurther, to assess the regulatory effect of FA + p-CA on aerobic glycolysis in CRC, the glucose consumption, ATP generation and lactate production were detected after the HCT-116 cells were treated with different concentration of FA + p-CA. The results showed that all the above indices were decreased A–C. AddiWe, therefore, considered whether lncRNA 495810 could regulate PKM2. The results showed that silencing lncRNA 495810 expression declined, whereas ectopic lncRNA 495810 expression enhanced the expression of PKM2. The PKM2 expression was positively correlated with lncRNA 495810. We therefore hypothesized that FA + p-CA inhibit aerobic glycolysis via the lncRNA 495810/PKM2 axis. To test our hypothesis, the HT-29 and HCT116 cells were transfected with 495810 and treated with FA + p-CA. The results showed that lncRNA 495810 knockdown enhanced and overexpression abrogated the inhibitory effect of FA + p-CA on PKM2 A–D. PhenRecently, extensive attention has been paid to the search for efficient and non-toxic anti-CRC drugs or functional molecules from natural products ,22,23. NMetabolic remodeling is one of the malignant features of tumor cells . TherefoAbnormal expression and function of lncRNA plays a key role in regulating tumor glucose metabolism remodeling ,32,33. IFerulic acid (FA) and p-coumaric acid (p-CA) were purchased from Victory Biological Technology Co., Ltd. . The content ratio of p-CA and FA in the mix was 1:1.13 . DMEM/F1n = 5 in each group): control group; AOM/DSS group; intervention group with different concentrations of FA + p-CA   were purchased from Beijing Weitahe Labor Animal Technology Co., Ltd. . All mice were raised in SPF cages in the laboratory at the Animal Service Center of China Institute of Radiation Protection. Animal experiments were approved by the National Institutes of Health and the Animal Experiment Ethics Committee of Shanxi University  under the principles of laboratory animal protection. The mice were divided into five groups (0 mg/kg) D. The co2.The colorectal cancer cell lines HT-29, HCT116 and SW620 were obtained from the Chinese Type Culture Collection . The cells were cultured in DMEM/F12 medium containing 10% FBS and placed in an incubator at 37 °C with 5% CODifferent colon cancer cells were seeded into 96-well plates, and after the cells were treated with different doses of FA + p-CA  for 48 h, 10 μL MTT was added for 4 h, and the OD value at 570 nm was detected.The cells were inoculated into the 6-well plate, and the culture medium and cells were collected after treating with FA + p-CA. The medium was used to determine the contents of glucose and lactate, and the cells were used to determine the production of ATP. The methods of measuring glucose, ATP, and lactate followed the manufacturer′s instructions.The total protein was extracted by WBIP, and the protein concentration was measured by BCA assay. The equal amount of protein was separated by SDS-PAGE and transferred to PVDF membrane. The membrane was blocked for 40 min at room temperature with 5% skim milk and incubated with different primary antibodies overnight for 4 °C. Then, the membrane was incubated with rabbit/mouse second antibody at room temperature for 2 h. Finally, the change of protein expression was observed by exposure.w/v = 1:7), and then ground with TissueLyser at 60 Hz for 2 min at 4 °C, then put on ice for 30 min. Centrifuge the lysate at 4 °C at 12,000 rpm for 15 min. The supernatant collected for protein concentration was determined with BCA detection kit. Next, the protein sample method is analyzed by Western blotting as described above.The mice intestinal tissue samples were taken from −80 °C, dissolved in a lysis buffer containing 1 mM PMSF  intervention were detected by RNA-seq. The LncRNA expression was accurately tested and corrected by Fisher and FDR values. The RNA-seq was conducted by Novogene Co., Ltd. .The total RNA was extracted from cells with Trizol reagent, and 500 ng RNA was reverse-transcribed using EasyScript First-Strand cDNA Synthesis SuperMix. The LncRNA and mRNA levels were quantified using qRT-PCR. GAPDH mRNA was used as the control. The primers used are listed in For the determination of siRNA transfection, the cells were inoculated into 6-well plates, and 10 nM siRNA was transfected with HiPerFect Transfection Reagent for 48 h according to the manufacturer′s scheme. For transient plasmid transfection, the plasmid was transfected into colon cancer cells using Turbofect reagent according to the manufacturer′s instructions. At 48 h after transfection, the cells were collected for subsequent analysis or determination. The siRNA sequences were as follows: lncRNA 495810-homo-112 .t-test, the values of p < 0.05 indicated that there was a significant difference, while p < 0.01 were considered that the difference was highly significant compared with control.The data were presented as the mean ± standard deviation of three independent experiments (Mean ± SD), data error bar indicated standard deviation. Single factor analysis of variance (ANOVA) was used for comparisons of means of ≥3 groups. Single variable comparisons were performed by Student′s In summary, we found that FA + p-CA exert the anti-tumor activity in CRC cells and AOM/DSS CRC mice. Moreover, FA + p-CA reverse the aerobic glycolysis through the lncRNA 495810/PKM2 cascade . Collect


Based on these results, the EWP-chitosan double-layer emulsion was prepared through the EWP-GPs being treated with 240 W ultrasound, TGase, and chitosan in this study. When the double-layer emulsion had 0.6% (v/v) chitosan, the zeta potential of the double-layer emulsion was −1.1 mV and the double-layer emulsion had a small particle size (56.87 µm). The creaming index of double-layer emulsion at 0.6% (v/v) chitosan was 16.3% and the droplets were dispersed uniformly. According to the rheological results, the storage modulus (G′) was larger than the loss modulus (G″) in the whole frequency, indicating the formation of an elastic gel network structure in the emulsion. It is hoped to develop a novel food-grade stabilizer and a stable double-layer emulsion, providing new environment-friendly processing in hen egg products and delivery systems.Egg-white protein has an abundance of hydrophobic amino acids and could be a potential emulsifier after modification. Here, egg-white protein was modified via ultrasonic and transglutaminase treatments to destroy the globular structure. The egg-white protein gel particles (EWP-GPs) were prepared and then a novel highly stable EWP-chitosan double-layer emulsion was constructed. When ultrasonic treatment was applied at 240 W and TGase (20 U/g EWP) treatment, the EWP-GPs had a low particle size and good emulsification performance. The particle size of EWP-GPs was a minimum of 287 nm, and the polymer dispersity index (PDI) was 0.41. The three-phase contact angle (θ Then, the W1/O emulsion was dispersed in the external aqueous phase (W2). It was reported that a W1/O/W2 emulsion should be stabilized with two kinds of emulsifiers (lipophilic to W1/O and hydrophilic to O/W2) to avoid the migration of the internal and external phases [Emulsion is a common material for delivering bioactive substances in food, pharmacy, and cosmetics , while swo steps . The firl phases . Additiol phases . Thus, Pl phases ,8,9. Fool phases . Among tl phases .In recent years, many protein particles have been used to stabilize Pickering emulsions, such as whey protein isolate  and zeinChitosan, a natural polysaccharide, is the by-product of chitin deacetylation . It has p < 0.05), and the PDI was at a minimum of 0.32. The PDI of EWP-GPs with 360 W ultrasonic treatment was significantly lower than the EWP-GPs without ultrasonic treatment (p < 0.05), indicating that 360 W ultrasonic treatment could improve the dispersion and stability of EWP-GPs. This phenomenon might be attributable to the mechanical shear produced by the ultrasonic treatment [The polymer dispersity index (PDI) was used to describe the molecular weight distribution of polymers . As showreatment . A studyreatment . Overallo/w), which could evaluate the adsorption efficiency of the emulsion. When the θo/w was less than 90°, the particles showed a good hydrophilicity. When the θo/w was more than 90°, the particles were inclined to show a good hydrophobicity. When the θo/w was 90°, the particles had the strongest absorption and could prevent the aggregation of droplets [o/w of EWP-GPs without any treatment was 52°: this indicated that the hydrophobicity of the particle was weak. The θo/w of EWP-GPs with 600 W ultrasonic treatment reached a peak (67.5°). These results indicated that the ultrasonic treatment could improve the hydrophobicity of EWP-GPs and that the EWP-GPs were partially wetted. It could be inferred that ultrasonic treatment caused the exposure of the protein chains and hydrophobic groups. A study suggested that ultrasonic power could destroy the non-covalent interaction of the whey proteins’ particles and enhance the hydrophobicity of particles [o/w of EWP-GPs with 240 W ultrasonic and TGase treatment was the largest (79.6°). This result showed that ultrasonic and TGase treatment could improve the hydrophile and lipophile of EWP-GPs. Transglutaminase could catalyze the formation of covalent bonds between lysine and glutamic acid in protein amino acids [As a Pickering particle, the particle should be insoluble but partially wetted by the continuous and dispersed phases. Partial wettability of particles contributed to sufficient interfacial adsorption efficiency. A three-phase contact angle of particles was the angle formed at the three-phase boundary where the solid particles, the continuous phase, and the dispersed phase intersect. The wettability of EWP-GPs could be evaluated by the three-phase contact angle . Egg-white protein contains a variety of proteins, and the isoelectric point is between 4 and 5 . Therefop < 0.001) compared with the EWP-GPs with 240 W ultrasonic treatment. It revealed that ultrasonic and TGase treatment could improve the emulsification of EWP-GPs. Ultrasonic and enzymatic treatment could increase the reaction sites and improve the emulsification of particles [Emulsification indicates the ability of a protein to adsorb at the oil–water interface. As articles ,35. The articles . The EWPp < 0.05). It was found that SDS, Urea, and DTT could destroy the hydrophobic interaction, hydrogen bond, and disulfide bond inside the particles, respectively [As ectively . TherefoIn conclusion, when the EWP-GPs were treated with 240 W ultrasound and TGase, the particles had small particles and good emulsification, which was suitable to prepare highly stable double-layer emulsions. In addition, when the pH value was 6, the EWP-GPs and chitosan were suitable to prepare the double-layer emulsion.p > 0.05). This might be due to the saturation of chitosan adsorption on the surface of the emulsion when the concentration of chitosan exceeded 0.8%. When the concentration of chitosan was less than 0.6%, chitosan molecules were adsorbed between the droplets due to electrostatic interaction. A study also found that electrostatic interaction between egg-white protein and κ-Carrageenan led to an increase in bridging flocculation and particle size [As cle size .As was shown in 2 to 103 s−1, indicating that the emulsions exhibited ideal fluid characteristics [The apparent shear viscosity of three emulsions decreased with an increase in shear rate a, indicaeristics .Storage modulus (G′) represents the elastic behavior of the emulsion network structure. The loss modulus (G″) represents the viscous behavior of the emulsion network structure. The hen eggs were purchased from the Beijing CP Egg Industry Co., Ltd. . Soybean oil  was purchased from a local market . Glutamine transaminase , chitosan, a Coomassie Brilliant Blue kit, Sodium Dodecyl Sulfate (SDS), Dithiothreitol (DTT), and ANS were purchased from Yuanye Bio-Technology Co., Ltd. .The egg-white protein gel particles (EWP-GPs) were prepared according to a previous method . The eggIn accordance with a previous method  and sligo/w) which was determined by using a VCA Optima system . The fresh EWP-GPs were diluted to 0.1% and dropped onto the mica sheet. After drying at 25 °C for 16 h, the water droplets (2 µL) were added to the composite particles to determine the θo/w. The θo/w was fitted to the Laplace-Young equation [In accordance with a previous method  with sliequation . Each saw/v) SDS. The absorbance of the emulsion at 500 nm was represented by the emulsification of the EWP-GPs.In accordance with a previous method  with sliw/w) SDS, and 30 mmol/L DTT). The control group was not treated with any protein denaturant . After 10 min of reaction, the EWP-GP solution was diluted to 0.1% (w/w) to remain in the linear region of absorbance. Each sample was measured at least three times in parallel, and the average value was taken. The transmittance (T) was calculated by the following formula:A represented the absorbance of the solution and T represented the transmittance of the solution.The intermolecular force of EWP-GPs was determined by measuring the absorbance of the solution at the wavelength of 600 nm at 25 °C with a UV/VIS spectrophotometer  INSTRUMENT CO., LTD., China) via a previous means  with mod1/O emulsion) was prepared with EWP-GP dispersion (4 wt.% EWP-GPs) and 40% (v/v) soybean oil, and the mixture was dispersed via a high-speed homogenizer at 15000 rpm for 2 min. Secondly, 0.2%, 0.4%, 0.6%, 0.8% and 1.0% (v/v) chitosan solutions (W2) were added to the Pickering emulsion, respectively. The mixture was dispersed via a high-speed homogenizer at 15,000 rpm for 2 min, and then egg-white protein-chitosan double-layer emulsion was obtained.In accordance with the previous study with slight modifications, the double-layer emulsion was prepared in two steps . FirstlyThe method to detect the particle size and zeta potential of the double-layer emulsion was the same as the method in In accordance with the reported means  with modHs) and the total height of emulsions (Ht) were recorded at fixed intervals. The experiment was carried out at 25 °C and the percentage of CI (%) was calculated using the following equation:Hs represented the height of the serum phase and Ht represented the total height of emulsions.The creaming index was measured via a reported method  with modThe rheology of the double-layer emulsions was performed with a Discovery DHR-2 Rheometer  by a previous means  with modp-values < 0.05 were considered as statistically significant.Data were presented as mean ± standard deviation (SD) of three independent experiments. The statistical analysis was assessed using one-way analysis of ANOVA with Duncan’s multiple range test. In this study, the EWP-GPs and EWP–chitosan double-layer emulsion were successfully prepared. The results showed that ultrasonic treatment combined with TGase treatment could significantly improve the surface hydrophobicity and stability of EWP-GPs. The optimal EWP-GPs had a smaller particle size, higher dispersity, better emulsification, and good wettability. The intermolecular force results showed that the hydrogen bond was the main force to maintain the stability of EWP-GPs. Based on these results, the EWP-chitosan double-layer emulsion was prepared and characterized. The double-layer emulsion had a small particle size and the droplets were dispersed uniformly, suggesting high stability. The rheological properties of the double-layer emulsion indicated that the gel network structure dominated by elasticity was formed in the emulsion. The prepared EWP-chitosan double-layer emulsion performed with high stability and good creaming stability. In a further study, we could study the delivery functions of the EWP-chitosan emulsion. It could provide a novel food-grade delivery system for bioactive substances, such as β-carotene and polyphenol.